ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1992, p. 942-948
Vol. 36, No. 5
0066-4804/92/050942-07$02.00/0 Copyright ©) 1992, American Society for Microbiology
Inhibitory Potency of Quinolone Antibacterial Agents against Cytochrome P450IA2 Activity In Vivo and In Vitro U. FUHR,1* E.-M. ANDERS,' G. MAHR,2 F. SORGEL,2 AND A. H. STAIB' Department of Clinical Phanmacology, University Hospital Frankfurt, 6000 Frankfiurt am Main 70,1 and Institute for Biomedical and Pharmaceutical Research, 8501 Niirnberg-Heroldsberg, Federal Republic of Germany
Received 3 September 1991/Accepted 17 February 1992
Inhibition of cytochrome P450OL2 activity is an important adverse effect of quinolone antibacterial agents. It results in a prolonged half-life for some drugs that are coadministered with quinolones, such as theophylline. The objective of the study described here was to define the parameters for quantifying the inhibitory potencies of quinolones against cytochrome P4501A2 in vivo and in vitro and to investigate the relationship between the results of both approaches. Cytochrome P450IA2 activity in vitro was measured by using the 3-demethylation rate of caffeine (500 FM) in human liver microsomes. The inhibitory potency of a quinolone in vitro was determined by calculating the decrease in the activity of cytochrome P450IA2 caused by addition of the quinolone (500 ,uM) into the incubation medium. The mean values (percent reduction of activity without quinolone) were as follows: enoxacin, 74.9%Yo; ciprofloxacin, 70.4%; nalidixic acid, 66.6%; pipemidic acid, 59.3%; norfloxacin, 55.7%; lomefloxacin, 23.4%; pefloxacin, 22.01%; amifloxacin, 21.4%; difloxacin, 21.3%; ofloxacin, 11.8%; temafloxacin, 10.01%; fleroxacin, no effect. The inhibitory potency of a quinolone in vivo was defined by a dose- and bioavailablity-normalized parameter calculated from changes of the elimination half-life of theophylline and/or caffeine reported in previously published studies. Taking the pharmacokinetics of the quinolones into account, it was possible to differentiate between substances with and without clinically relevant inhibitory effects by using results of in vitro investigations. The in vitro test described here may help to qualitatively predict the relevant drug interactions between quinolones and methylxanthines that occur during therapy.
The fluoroquinolones have proven to be very effective antibacterial agents (2, 20, 23). Several new agents have been developed over the past decade. Their efficacies in controlling bacterial infections have resulted in the widespread use of these drugs. Another important biological effect of this drug class concerns the metabolism of several unrelated pharmaceutical agents, resulting in occasional adverse reactions. It has been reported that some of the quinolone antibacterial agents cause a reduced velocity of caffeine and theophylline degradation in vivo (see below) (for a review, see reference 10) and in vitro (14, 40). Furthermore, reductions in the antipyrine metabolism rate and the R-warfarin oxidation rate were observed in humans when quinolones were coadministered with these drugs (23, 24, 46). In investigations with human liver microsomes, the mechanism of this interaction was determined for ofloxacin, ciprofloxacin, enoxacin, lomefloxacin, and pipemidic acid. A competitive-type inhibition of P450LA2 activity by all of these compounds was found (14). This cytochrome P450 isoform is primarily responsible for the first steps of both caffeine and theophylline metabolism in the liver, as shown in investigations with specific antibodies (5, 37) and genetically engineered cells that express single cytochrome P450 isoforms (13). The main metabolic pathway of caffeine in all systems tested, i.e., 3-demethylation, is mediated almost exclusively by cytochrome P450IA2 (5) and, therefore, may be used as a specific probe for P450LA2 activity (13). Serious incidents (theophylline intoxication), including death, have occurred because of this drug interaction (3, 25). Thus, the objective of the present study was to define the *
parameters for quantifying the inhibitory potencies of quinolones against cytochrome P450IA2 in vivo and in vitro and to investigate the relationship between in vitro and in vivo effects. This is of considerable interest in light of the abundance of new quinolones. The probability of encountering these adverse drug reactions should be considered when comparing the therapeutic values of otherwise similar quinolones.
MATERIALS AND METHODS Quinolones and other chemicals. The following quinolones were supplied by the indicated companies: ofioxacin (Hoechst, Frankfurt, Federal Republic of Germany), lomefloxacin (Searle, Dreieichenhain, Federal Republic of Germany), pipemidic acid (Madaus, Cologne, Federal Republic of Germany), ciprofloxacin (Bayer, Wuppertal, Federal Republic of Germany), enoxacin (Gddecke, Freiburg, Federal Republic of Germany), difloxacin and temafloxacin (Abbott, North Chicago, Ill.), pefloxacin (Rhone-Poulenc, Antony, France), norfloxacin (Merck Sharp & Dohme, Rahway, N.J.), nalidixic acid and amifloxacin (Winthrop, Norderstedt, Federal Republic of Germany), and fleroxacin (Kyorin, Tokyo, Japan). The following methylxanthines were obtained from the indicated suppliers: caffeine (Serva, Heidelberg, Federal Republic of Germany), 1,7-dimethylxanthine (1,7-DMX; Aldrich, Milwaukee, Wis.), and hydroxypropyltheophylline (Fluka, Buchs, Switzerland). NADP (98%), glucose 6-phosphate, and glucose 6-phosphate-dehydrogenase (from yeast, purity grade II, 140 U/ml) were purchased from Boehringer (Mannheim, Federal Republic of Germany). Acetonitrile, methanol, and tetrahydrofuran were chromatography-grade products from Merck (Darmstadt, Federal Republic of Ger-
Corresponding author. 942
QUINOLONE-METHYLXANTHINE INTERACTION
VOL. 36, 1992
many). All other chemicals (analytical grade) were purchased from Merck. In vitro data. The effect of quinolones on the 3-demethylation of caffeine in human liver microsomes was used to measure the inhibitory activity against cytochrome P450IA2. (i) Tissue preparation. Donors of liver samples, the preparation of human liver microsomes, and characterization methods were the same as described previously (14). (ii) Incubations. Incubations were carried out in 0.1 M KH2PO4 buffer (pH 7.4), in which all compounds were dissolved. Since the solubilities of some of the quinolones in this buffer were poor, stock solutions of quinolones were prepared by dissolving about 5 ,umol in 0.2 ml of 0.1 M NaOH. An adequate volume of this solution (1.5 ,umol of inhibitor) was immediately added to buffer containing 0.023 mM glucose 6-phosphate. Together with 0.1 M HCl (volume equal to that of the inhibitor solution), a final volume of 1.5 ml with a final inhibitor concentration of 1 mM was obtained for the stock solutions. The quinolone to be tested was added to a buffer solution containing about 2 mg of microsomal protein per ml, caffeine, and an NADPH-generating system (for details, see reference 14). The incubation time was 15 min. Caffeine concentrations of 500 ,uM were chosen to test the inhibitory effect of a quinolone derivative. A quinolone concentration of 500 p,M was used. As a control, incubations in the absence of a quinolone but with otherwise identical conditions were carried out. Four independent values were obtained during separate incubations by using human liver microsomes from four donors; each value was derived from duplicate incubations with and without an inhibitor. The choice of concentrations used in the in vitro test system depended on the analytical conditions. Both substrate and inhibitor concentrations exceeded those that occur in vivo by about 1 order of magnitude. This was necessary because the activity of caffeine metabolism in vitro is low (13), and neither metabolite concentrations nor inhibitory effects could be measured at lower concentrations in vitro. Although it would be possible to extrapolate the effects under chosen in vitro conditions to any substrate and/or inhibitor concentration, such a procedure was not helpful in this study, since the concentrations of both caffeine and the quinolones at the active site of the enzyme were unknown. (iii) Determination of 1,7-DMX. 1,7-DMX was assayed by high-pressure liquid chromatography (14). 1,7-DMX peak heights relative to those of the internal standard showed a linear correlation (r > 0.998) with spiked concentrations in the concentration range from 250 to 100,000 nM. The coefficient of variation of the assay was 7.1% at 250 nM and below 3% at concentrations greater than 1,000 nM. HPLC run times were prolonged by up to 1 h to avoid peak superposition of methylxanthines by the quinolones from a previous run. (iv) Evaluation of in vitro data. The inhibitory effect (Ein vitro) of a compound on microsomes from each donor was determined as cytochrome P450IA2 activity in the presence of this compound relative to that in its absence. This effect is given as a mean + standard deviation for the four microsome specimens. Inhibitory potency (Pin vitro) was = 1 calculated as the mean reduction of activity (Pin vitro
Ein vitro).
In vivo data. (i) Inhibitory effects of quinolones. The effects of 10 quinolone antibacterial agents on methylxanthine metabolism in vivo were summarized from previously published data, including our own investigations (4, 7, 9, 11,
943
16-18, 22, 26, 28, 31, 32, 35, 36, 38, 39, 41-44, 47, 48). Those studies dealt with changes of theophylline and caffeine kinetics caused by the concomitant repeated application of quinolones in therapeutic doses. (ii) Quinolone pharmacokinetics. The inhibitory effects of the quinolones are expected to be related to their concentrations at the cytochrome P450IA2 site. These concentrations following oral administration are unknown, and even serum quinolone concentrations in interaction studies have been measured only occasionally. The existing data obtained from studies of quinolone pharmacokinetics are not sufficient to enable a reliable estimation of the quinolone concentrations obtained in interaction studies. Thus, only the bioavailability and amount of the quinolone given were taken into account in evaluating inhibitory properties in vivo. (iii) Evaluation of in vivo data. The extent of inhibition reported in vivo was expressed as follows using mean values for the half-life (or reciprocal clearance): Ein vivo = 1 (half-life of caffeine or of theophylline when administered alone)/(half-life of caffeine or of theophylline during quinolone coadministration) (equation 1). An in vivo inhibitory effect (Ein i,. of 0 was assumed when a decreased half-life during quinolone coadministration was reported. From these reported effects, we calculated a standard inhibitory potency (Pin vivo) which represents a dose- and bioavailabilitynormalized parameter for each quinolone. To that end, the dose-dependent effects of all compounds were fitted to the log-linear sigmoid dose-effect model described in the following equation by using nonlinear regression analysis (19): Ein vivo = (brax dosen)/(ED50' + doses) (equation 2), where is the maximal effect, and ED50 is the dose that gives bma 50% of the maximal effect. The value of bma used in this equation was 1 (i.e., theoretical prolongation of methylxanthine half-life to infinity); the dose term describes the singledose size of the drug that was coadministered, which was given once to four times daily (usually twice daily). The exponent n was fixed at 1, because the inclusion of this variable in a nonlinear regression analysis in most cases was not favored by the Akaike model discrimination test (34). All datum points were given the same weight in the analysis. To allow for differences in quinolone doses in interaction studies, a dose of 1 mmol was chosen for all quinolones to compare the effects between substances. The bioavailability of a quinolone was taken into account by dividing the 1-mmol dose by the absolute oral bioavailability of the substance. For ciprofloxacin a value of 0.60 for oral bioavailability was used (20), for norfloxacin the value was 0.35 (1), and for all other compounds, it was 1.00 (2, 30, 33). Thus, the effect of a dose resulting in a bioavailable drug amount of 1 mmol was calculated from the fitted curves and was used to describe the Pin vivo of a quinolone. These doses (e.g., enoxacin, 321 mg; fleroxacin, 406 mg; ciprofloxacin, 553 mg) are within the range of therapeutic doses for most quinolones
(2, 30, 33). RESULTS Table 1 presents the Ein vitro values of the quinolones expressed as the remaining activity of P450IA2 for caffeine 3-demethylation when a quinolone (500 ,uM) was added to the incubation relative to the control activity in the absence of an inhibitor. Pin vitro values are also, given in Table 1. The coefficient of variation for Ein vitro between donors was small (about 10%). Enoxacin was the most effective inhibitor of caffeine 3-demethylation tested in vitro, whereas fleroxacin showed no inhibitory activity.
944
FUHR ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Pin vitro of quinolones by using caffeine 3-demethylation in human liver microsomes Compound
Enoxacin Ciprofloxacin Nalidixic acid Pipemidic acid Norfloxacin Lomefloxacin Pefloxacin Amifloxacin Difloxacin Ofloxacin Temafloxacin Fleroxacin
Ein *,tro determined with
liver microsomes from four donors (mean + SD)'
0.251 0.306 0.334 0.407 0.443 0.766 0.780 0.786 0.787 0.892 0.900 1.044
± ± ± ± ± ±
+ ± ± + + ±
0.017 0.067 0.056 0.028 0.089 0.071 0.048 0.038 0.073 0.099 0.045 0.142
Pin vitrob 0.749 0.694 0.666 0.593 0.557 0.234 0.220 0.214 0.213 0.108 0.100 0.000
a Expressed as the remaining activity of P450LA2 when a quinolone was added to the incubation as a fraction of the control activity. b Relative reduction of enzyme activity.
The Ein vivo values of the quinolones were assessed from the interaction studies with theophylline or caffeine reported above. The dose-dependent extent of the effects of the quinolones on caffeine metabolism were similar to those on theophylline degradation for all quinolones for which data from interaction studies were available. Whenever any effect of quinolones was found, higher doses caused a greater prolongation of the half-life, resulting in a higher Ein viv. (Table 2). Although study designs and study subjects differed widely, only small differences between the observed and the calculated effects were found for each quinolone (Table 2). Parameters estimated by using equation 2 for each quinolone are given in Table 3. As an example, the relation between the dose and inhibitory effect in vivo is shown for enoxacin and ciprofloxacin (Fig. 1). The extent of methylxanthine metabolism inhibition to be expected from the repeated application of a dose that results in a bioavailable quinolone amount of 1 mmol (i.e., 232 to 901 mg) (Pin viv.) was derived from reported values (see above) and is given in Fig. 2. The relationship of inhibitory potency in both test systems is shown by plotting Pin vv versus Pin vitro (Fig. 3). There was no linear relationship present for the substances tested here. However, by separating quinolone groups with high and low inhibitory potencies in vivo and in vitro by mean values, it was shown that pronounced decreases in cytochrome P450IA2 activity (Pin vitro, >0.39) in microsomes usually correspond to a marked prolongation of the methylxanthine half-life in vivo (Pin vivo, >0.22) and that minor effects in vitro were related only to slight changes in the pharmacokinetic parameters in volunteers. Exceptions were nalidixic acid, which had a high inhibitory effect in vitro but not in vivo, and pefloxacin, for which the effect in vivo was more pronounced than that in vitro. DISCUSSION
All quinolones tested (with the exception of fleroxacin) showed an inhibitory effect on caffeine 3-demethylation under the in vitro conditions used in this study. This effect was most pronounced for enoxacin and ciprofloxacin. A possible modification of the in vitro effects because of degradation of the quinolones has been tested for ciprofloxacin (12). Reduction in the concentration of intact ciproflox-
acin was not detectable as a result of incubation. The concentration of the Ml metabolite (cleavage of piperazinyl substituent) of ciprofloxacin was less than 0.3% of the ciprofloxacin concentration, and other metabolites were below the limit of detection. It has been reported that some quinolones (i.e., enoxacin, ciprofloxacin, ofloxacin, lomefloxacin, and pipemidic acid [14, 29]) exert a competitive type of inhibition on caffeine 3-demethylation. The structures of the substances tested previously (14, 29) cover the range of structures of those of the compounds included in the present investigation. Therefore, it is safe to conclude that all congeners used in this study exerted their inhibitory effects by binding to the active site of the enzyme, resulting in a competitive type of inhibition. A competitive inhibition is described by equation 3: v = (Vmax [S])/{[S] + Km (1 + [I]lKi)}, where v is the metabolite formation rate, Vm. is the maximum metabolite formation rate possible, [S] is the substrate concentration, Km is the Michaelis-Menten constant, [I] is the inhibitor concentration, and Ki is the inhibitor constant. By using equation 3, Ki values (or the 50% inhibitory concentration) can be calculated from a single substrate and inhibitor concentration since Km and Vm. values have previously been determined for this reaction in the same microsome samples (14). Therefore, the information given by the percent inhibition obtained in the present study is equivalent to that provided by the Ki or 50% inhibitory concentration parameters, which are used more often. However, the percentages were not transformed to avoid a multiplication of the experimental error. The low standard deviation between donors provides further evidence that P450 isozymes other than P450IA2 (or isoforms coordinately regulated with P450IA2) that are not susceptible to inhibition do not take part in caffeine metabolism (5). Cytochrome P450IA2 has been reported to be responsible for caffeine 3-demethylation, which is the major caffeine degradation step in humans (5). There is evidence that this isozyme also takes part in 1- and 7-demethylation of caffeine and mediates the main portions of the theophylline demethylation and hydroxylation pathways (although other cytochrome P450 isoforms may also play a minor part in primary theophylline metabolism [37]). Additionally, similar Km values for all primary metabolic steps of theophylline and caffeine have been reported (6, 14, 15, 21). Thus, it is not surprising that inhibition of P450IA2 activity by the concomitant application of quinolones results in a similar degree of half-life prolongation and/or clearance reduction for both caffeine and theophylline (10). This enables the pooling of in vivo inhibition data for both methylxanthines. The association between the increasing half-lives of the methylxanthines with higher doses of the inhibitory quinolones (16, 38) may readily be explained by the competitive type of inhibition that has been observed in vitro (14). To quantify the dose dependency of these inhibitory effects, we derived a dose- and bioavailability-normalized inhibitory potency (Pin vivo) for each quinolone for which interaction studies with methylxanthines have been reported previously. This defined a mean increase in the methylxanthine half-life that resulted from repeated, oral coadministration of a quinolone dose representing a bioavailable amount of 1 mmol. Pin vivo showed only minor variations within each of the compounds examined in this study, and therefore, it may be a good parameter to use to describe the inhibitory potency of quinolones. Nevertheless, each of the interaction studies
945
QUINOLONE-METHYLXANTHINE INTERACTION
VOL. 36, 1992
TABLE 2. Effects of quinolone antibacterial agents on methylxanthine elimination in vivo Quinolone administration Compound Compound
Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Pipemidic acid Pipemidic acid Pipemidic acid Pefloxacin Pefloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin
Ciprofloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Fleroxacin Fleroxacin Lomefloxacin Lomefloxacin Lomefloxacin Nalidixic acid Nalidixic acid Ofloxacin Ofloxacin Ofloxacin Ofloxacin Ofloxacin Temafloxacin
~
Compound (days) (days)'
25 mg b.i.d., 7 100 mg b.i.d., 7 400 mg b.i.d., 7 200 mg b.i.d., 4 100 mg b.i.d., 4 200 mg b.i.d., 4 400 mg b.i.d., 6 400 mg b.i.d., 7 500 mg b.i.d., 4 400 mg b.i.d., 4 400 mg b.i.d., 5 400 mg b.i.d., 4 800 mg b.i.d., 1.5 400 mg b.i.d., 6 400 mg b.i.d., 4 100 mg b.i.d., 4 250 mg b.i.d., 4 500 mg b.i.d., 4 750 mg b.i.d., 7 500 mg b.i.d., 7 750 mg b.i.d., 6 500 mg b.i.d., 7 500 mg b.i.d., 6 750 mg b.i.d., 1.5 400 mg b.i.d., 4 400 mg b.i.d., 4 200 mg t.i.d., 3 400 mg b.i.d., 6 800 mg b.i.d., 1.5 400 mg b.i.d., 7 400 mg b.i.d., 4 200 mg b.i.d., 5 400 mg b.i.d., 5 200 mg b.i.d., 5 400 mg b.i.d., 7 400 mg o.d., 5 500 mg b.i.d., 6 500 mg q.i.d., 7 200 mg b.i.d., 4 400 mg b.i.d., 6 200 mg t.i.d., 3 200 mg b.i.d., 5 200 mg b.i.d., 4 400 mg b.i.d., 7
Methylxanthine administration Dosing, duration Compound
Dosing, duration (days)
Theophylline Theophylline Theophylline Theophylline Caffeine Caffeine Theophylline Theophylline Caffeine Caffeine Theophylline Caffeine Caffeine Theophylline Caffeine Caffeine Caffeine Caffeine Theophylline Theophylline Theophylline Theophylline Theophylline Caffeine Theophylline Caffeine Theophylline Theophylline Caffeine Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Caffeine Theophylline Theophylline Theophylline Theophylline Theophylline
200 mg, single dose 200 mg, single dose 200 mg, single dose 4 mg/kg, single dose 230 mg, single dose 230 mg, single dose 300-600 mg b.i.d. 146 mg b.i.d., 5 230 mg, single dose 183 mg o.d., 4 260 mg, single dose 230 mg, single dose 350 mg, single dose 300-600 mg b.i.d. 183 mg o.d., 4 230 mg, single dose 230 mg, single dose 230 mg, single dose 4 mg/kg, single dose 125 mg t.i.d., 7 200 mg b.i.d., 20 5 mg/kg, single dose 300-600 mg b.i.d. 100 mg, single dose 4 mg/kg, single dose 230 mg, single dose 183 mg, single dose 4 mg/kg, single dose 350 mg, single dose 5 mg/kg, single dose 142 mg t.i.d., 4 200 mg b.i.d., 9 141 mg b.i.d., 5 200 mg b.i.d., 10 125 mg t.i.d., 7 260 mg, single dose 300-600 mg b.i.d. 5 mg/kg, single dose 230 mg, single dose 300-600 mg b.i.d. 183 mg, single dose 200 mg t.i.d., 5 4 mg/kg, single dose 146 mg b.i.d., 5
E
iEn oba
eserved inE esimate
Reference
0.132 0.056 -0.247 -0.034 0.140 0.020 -0.085 -0.047 -0.024 0.136 -0.042 0.079 -0.039 -0.100 0.099 -0.005 -0.011 -0.035 -0.066 0.022 -0.052 0.048 0.066 0.040 -0.052 -0.002 0.068 0.008 -0.063 0.044 0.066 -0.033 0.017 -0.022 -0.026 0.037 -0.048 0.048 -0.031 -0.043 0.018 0.021 0.072 0.000
38 38 38 47 16 16 33 28 16 26 44 16 7 33 26 16 16 16 32 36 41 35 33 17 9 16 39 18 7 35 4 43 42 22 36 44 33 35 16 33 39
E.
0.259 0.424 0.452 0.504 0.508 0.558 0.614 0.652 0.720 0.835 0.427 0.548 0.600 0.291 0.490 0.056 0.128 0.209 0.260 0.266 0.274 0.292 0.310 0.366 0.084 0.136 0.144 0.149 0.184 0.185 0.207 0.000 0.081 0.000 0.018 0.081 0.000 0.096 0.000 0.017 0.049 0.052 0.103 0.086
11 47 28
a For calculation of parameters, see text; an Ein ,v, of 0 corresponds to no increase in the methylxanthine half-life during quinolone coadministration; of 1 represents a theoretical half-life prolongation to infinity. b o.d., once daily; b.i.d., two times daily; t.i.d., three times daily; q.i.d., four times daily. C Dose per kilogram of body weight.
(see above) showed
a
high interindividual variation in the
Ein ^i, value which may have been due to the well-known
intraindividual variation of methylxanthine metabolism in the course of a study and/or to interindividual differences in the disposition of quinolones. Differences in the susceptibilities of individual P450IA2 cytochromes in vivo seem to be of minor importance, since the effects of the quinolones on the enzyme in vitro were very similar between donors (Table 1). Thus, it is possible to estimate mean changes in methylxanthine kinetics because of quinolone coadministration at a defined dose by using equation 2 and the parameters listed in Table 3. For example, coadministration of pefloxacin (200 mg twice daily) in patients with asthma who are taking theophylline is estimated to cause an Ein i, of 0.243. This corresponds to a mean prolongation in the theophylline
a
value
TABLE 3. Parameters estimated by equation 2 describing the dose-effect relationship of the quinolones and inhibitory activity on methylxanthine elimination in vivo Compound
Enoxacin .........
Ciprofloxacin .........
ED50 (mg)a
172
1,550
Nalidixic acid ......... 9,917 452 Pipemidic acid ......... Norfloxacin ......... 2,441 Lomefloxacin ......... 8,760 624 Pefloxacin ......... Ofloxacin ......... 6,258 Temafloxacin ......... 4,251 Fleroxacin ......... 5,898 a The ED50 is the oral dose (given twice daily in most cases) estimated to double the caffeine or theophylline half-life when one of the quinolones is coadministered with one of the methylxanthines.
946
FUHR ET AL.
ANTIMICROB. AGENTS CHEMOTHER. 1.0& values reported for ciprofloxacin o values reported for enoxacin - curves fitted using eq. 2
0
>
*n
0.6
/
-4-,
13
0
c
13
0.8-
0.4
0
~~~~~~~~~~A 0f
2:N 0
C~~~~~~~~~~~~~C
0.01
10
100
1000
oral quinolone dose size
1 E+04
(mg)
FIG. 1. Influence of enoxacin and of ciprofloxacin dose on P450IA2 activity in vivo. The dose on the x axis describes the size of a single oral quinolone dose which was applied repeatedly. Ei i,. iS 1 minus the quotient of the methylxanthine half-lives reported without and during concomitant quinolone application. lE + 04, io'.
half-life of 32%, which requires a mean dose reduction by the same percentage to maintain steady-state trough levels when an open one-compartment model for theophylline kinetics is applied. However, a corresponding dose adjustment based on mean inhibitory effects in individual patients with longterm theophylline therapy still requires control for possible adverse drug reactions and/or concentrations of drug in serum because of interindividual diversity (see above). For the majority of quinolones investigated, the inhibitory
effects observed under the chosen in vitro conditions were related to qualitatively similar effects in vivo. Enoxacin, pipemidic acid, norfloxacin, and ciprofloxacin caused marked decreases in caffeine metabolite formation rates in vitro (Pin vitro .0.39) and produced a significant prolongation of methylxanthine half-lives in most in vivo studies (Pin viva' .0.22). Temafloxacin, lomefloxacin, ofloxacin, and fleroxacin had only minor effects both in vivo and in vitro. The inhibitory effects of quinolones on methylxanthine metabolism in vivo are postulated to be the result of a mutually exclusive concurrence at the cytochrome P450IA2
1.0-i 0
1.0-
0.8c
0~
0.8-
0~
0.6a.
0)
*ENX 0.6-
0. 0.40.
0.4-
a)
*PEF
_.
-0c
0.2-
*PPA
*NOR * CIp 0.2-
0.0-
FLXT 0.0
FIG. 2. Inhibitory potency of various quinolones against methylxanthine metabolism in vivo. Pi, ,j is 1 minus the quotient of the methylxanthine half-lives estimated without and during repeated application of a quinolone dose resulting in a bioavailable drug amount of 1 mmol. Abbreviations: ENX, enoxacin; PPA, pipemidic acid; PEF, pefloxacin; NOR, norfloxacin; CIP, ciprofloxacin; TEF, temafloxacin; FLX, fleroxacin; OFL, ofloxacin; LFX, lomefloxacin; NLA, nalidixic acid.
0.0
*LFX
*NLA
0.2 0.4 0.6 0.8 inh. potency in hum. liver microsomes (P in vitro)
1.0
FIG. 3. Relationship of inhibitory potency of quinolones against cytochrome P450IA2 activity in vivo and in vitro. Lines represent mean values that were used to distinguish between substances with minor inhibitory activities in vivo (Pi iv,a,
Vol. 36, No. 5
0066-4804/92/050942-07$02.00/0 Copyright ©) 1992, American Society for Microbiology
Inhibitory Potency of Quinolone Antibacterial Agents against Cytochrome P450IA2 Activity In Vivo and In Vitro U. FUHR,1* E.-M. ANDERS,' G. MAHR,2 F. SORGEL,2 AND A. H. STAIB' Department of Clinical Phanmacology, University Hospital Frankfurt, 6000 Frankfiurt am Main 70,1 and Institute for Biomedical and Pharmaceutical Research, 8501 Niirnberg-Heroldsberg, Federal Republic of Germany
Received 3 September 1991/Accepted 17 February 1992
Inhibition of cytochrome P450OL2 activity is an important adverse effect of quinolone antibacterial agents. It results in a prolonged half-life for some drugs that are coadministered with quinolones, such as theophylline. The objective of the study described here was to define the parameters for quantifying the inhibitory potencies of quinolones against cytochrome P4501A2 in vivo and in vitro and to investigate the relationship between the results of both approaches. Cytochrome P450IA2 activity in vitro was measured by using the 3-demethylation rate of caffeine (500 FM) in human liver microsomes. The inhibitory potency of a quinolone in vitro was determined by calculating the decrease in the activity of cytochrome P450IA2 caused by addition of the quinolone (500 ,uM) into the incubation medium. The mean values (percent reduction of activity without quinolone) were as follows: enoxacin, 74.9%Yo; ciprofloxacin, 70.4%; nalidixic acid, 66.6%; pipemidic acid, 59.3%; norfloxacin, 55.7%; lomefloxacin, 23.4%; pefloxacin, 22.01%; amifloxacin, 21.4%; difloxacin, 21.3%; ofloxacin, 11.8%; temafloxacin, 10.01%; fleroxacin, no effect. The inhibitory potency of a quinolone in vivo was defined by a dose- and bioavailablity-normalized parameter calculated from changes of the elimination half-life of theophylline and/or caffeine reported in previously published studies. Taking the pharmacokinetics of the quinolones into account, it was possible to differentiate between substances with and without clinically relevant inhibitory effects by using results of in vitro investigations. The in vitro test described here may help to qualitatively predict the relevant drug interactions between quinolones and methylxanthines that occur during therapy.
The fluoroquinolones have proven to be very effective antibacterial agents (2, 20, 23). Several new agents have been developed over the past decade. Their efficacies in controlling bacterial infections have resulted in the widespread use of these drugs. Another important biological effect of this drug class concerns the metabolism of several unrelated pharmaceutical agents, resulting in occasional adverse reactions. It has been reported that some of the quinolone antibacterial agents cause a reduced velocity of caffeine and theophylline degradation in vivo (see below) (for a review, see reference 10) and in vitro (14, 40). Furthermore, reductions in the antipyrine metabolism rate and the R-warfarin oxidation rate were observed in humans when quinolones were coadministered with these drugs (23, 24, 46). In investigations with human liver microsomes, the mechanism of this interaction was determined for ofloxacin, ciprofloxacin, enoxacin, lomefloxacin, and pipemidic acid. A competitive-type inhibition of P450LA2 activity by all of these compounds was found (14). This cytochrome P450 isoform is primarily responsible for the first steps of both caffeine and theophylline metabolism in the liver, as shown in investigations with specific antibodies (5, 37) and genetically engineered cells that express single cytochrome P450 isoforms (13). The main metabolic pathway of caffeine in all systems tested, i.e., 3-demethylation, is mediated almost exclusively by cytochrome P450IA2 (5) and, therefore, may be used as a specific probe for P450LA2 activity (13). Serious incidents (theophylline intoxication), including death, have occurred because of this drug interaction (3, 25). Thus, the objective of the present study was to define the *
parameters for quantifying the inhibitory potencies of quinolones against cytochrome P450IA2 in vivo and in vitro and to investigate the relationship between in vitro and in vivo effects. This is of considerable interest in light of the abundance of new quinolones. The probability of encountering these adverse drug reactions should be considered when comparing the therapeutic values of otherwise similar quinolones.
MATERIALS AND METHODS Quinolones and other chemicals. The following quinolones were supplied by the indicated companies: ofioxacin (Hoechst, Frankfurt, Federal Republic of Germany), lomefloxacin (Searle, Dreieichenhain, Federal Republic of Germany), pipemidic acid (Madaus, Cologne, Federal Republic of Germany), ciprofloxacin (Bayer, Wuppertal, Federal Republic of Germany), enoxacin (Gddecke, Freiburg, Federal Republic of Germany), difloxacin and temafloxacin (Abbott, North Chicago, Ill.), pefloxacin (Rhone-Poulenc, Antony, France), norfloxacin (Merck Sharp & Dohme, Rahway, N.J.), nalidixic acid and amifloxacin (Winthrop, Norderstedt, Federal Republic of Germany), and fleroxacin (Kyorin, Tokyo, Japan). The following methylxanthines were obtained from the indicated suppliers: caffeine (Serva, Heidelberg, Federal Republic of Germany), 1,7-dimethylxanthine (1,7-DMX; Aldrich, Milwaukee, Wis.), and hydroxypropyltheophylline (Fluka, Buchs, Switzerland). NADP (98%), glucose 6-phosphate, and glucose 6-phosphate-dehydrogenase (from yeast, purity grade II, 140 U/ml) were purchased from Boehringer (Mannheim, Federal Republic of Germany). Acetonitrile, methanol, and tetrahydrofuran were chromatography-grade products from Merck (Darmstadt, Federal Republic of Ger-
Corresponding author. 942
QUINOLONE-METHYLXANTHINE INTERACTION
VOL. 36, 1992
many). All other chemicals (analytical grade) were purchased from Merck. In vitro data. The effect of quinolones on the 3-demethylation of caffeine in human liver microsomes was used to measure the inhibitory activity against cytochrome P450IA2. (i) Tissue preparation. Donors of liver samples, the preparation of human liver microsomes, and characterization methods were the same as described previously (14). (ii) Incubations. Incubations were carried out in 0.1 M KH2PO4 buffer (pH 7.4), in which all compounds were dissolved. Since the solubilities of some of the quinolones in this buffer were poor, stock solutions of quinolones were prepared by dissolving about 5 ,umol in 0.2 ml of 0.1 M NaOH. An adequate volume of this solution (1.5 ,umol of inhibitor) was immediately added to buffer containing 0.023 mM glucose 6-phosphate. Together with 0.1 M HCl (volume equal to that of the inhibitor solution), a final volume of 1.5 ml with a final inhibitor concentration of 1 mM was obtained for the stock solutions. The quinolone to be tested was added to a buffer solution containing about 2 mg of microsomal protein per ml, caffeine, and an NADPH-generating system (for details, see reference 14). The incubation time was 15 min. Caffeine concentrations of 500 ,uM were chosen to test the inhibitory effect of a quinolone derivative. A quinolone concentration of 500 p,M was used. As a control, incubations in the absence of a quinolone but with otherwise identical conditions were carried out. Four independent values were obtained during separate incubations by using human liver microsomes from four donors; each value was derived from duplicate incubations with and without an inhibitor. The choice of concentrations used in the in vitro test system depended on the analytical conditions. Both substrate and inhibitor concentrations exceeded those that occur in vivo by about 1 order of magnitude. This was necessary because the activity of caffeine metabolism in vitro is low (13), and neither metabolite concentrations nor inhibitory effects could be measured at lower concentrations in vitro. Although it would be possible to extrapolate the effects under chosen in vitro conditions to any substrate and/or inhibitor concentration, such a procedure was not helpful in this study, since the concentrations of both caffeine and the quinolones at the active site of the enzyme were unknown. (iii) Determination of 1,7-DMX. 1,7-DMX was assayed by high-pressure liquid chromatography (14). 1,7-DMX peak heights relative to those of the internal standard showed a linear correlation (r > 0.998) with spiked concentrations in the concentration range from 250 to 100,000 nM. The coefficient of variation of the assay was 7.1% at 250 nM and below 3% at concentrations greater than 1,000 nM. HPLC run times were prolonged by up to 1 h to avoid peak superposition of methylxanthines by the quinolones from a previous run. (iv) Evaluation of in vitro data. The inhibitory effect (Ein vitro) of a compound on microsomes from each donor was determined as cytochrome P450IA2 activity in the presence of this compound relative to that in its absence. This effect is given as a mean + standard deviation for the four microsome specimens. Inhibitory potency (Pin vitro) was = 1 calculated as the mean reduction of activity (Pin vitro
Ein vitro).
In vivo data. (i) Inhibitory effects of quinolones. The effects of 10 quinolone antibacterial agents on methylxanthine metabolism in vivo were summarized from previously published data, including our own investigations (4, 7, 9, 11,
943
16-18, 22, 26, 28, 31, 32, 35, 36, 38, 39, 41-44, 47, 48). Those studies dealt with changes of theophylline and caffeine kinetics caused by the concomitant repeated application of quinolones in therapeutic doses. (ii) Quinolone pharmacokinetics. The inhibitory effects of the quinolones are expected to be related to their concentrations at the cytochrome P450IA2 site. These concentrations following oral administration are unknown, and even serum quinolone concentrations in interaction studies have been measured only occasionally. The existing data obtained from studies of quinolone pharmacokinetics are not sufficient to enable a reliable estimation of the quinolone concentrations obtained in interaction studies. Thus, only the bioavailability and amount of the quinolone given were taken into account in evaluating inhibitory properties in vivo. (iii) Evaluation of in vivo data. The extent of inhibition reported in vivo was expressed as follows using mean values for the half-life (or reciprocal clearance): Ein vivo = 1 (half-life of caffeine or of theophylline when administered alone)/(half-life of caffeine or of theophylline during quinolone coadministration) (equation 1). An in vivo inhibitory effect (Ein i,. of 0 was assumed when a decreased half-life during quinolone coadministration was reported. From these reported effects, we calculated a standard inhibitory potency (Pin vivo) which represents a dose- and bioavailabilitynormalized parameter for each quinolone. To that end, the dose-dependent effects of all compounds were fitted to the log-linear sigmoid dose-effect model described in the following equation by using nonlinear regression analysis (19): Ein vivo = (brax dosen)/(ED50' + doses) (equation 2), where is the maximal effect, and ED50 is the dose that gives bma 50% of the maximal effect. The value of bma used in this equation was 1 (i.e., theoretical prolongation of methylxanthine half-life to infinity); the dose term describes the singledose size of the drug that was coadministered, which was given once to four times daily (usually twice daily). The exponent n was fixed at 1, because the inclusion of this variable in a nonlinear regression analysis in most cases was not favored by the Akaike model discrimination test (34). All datum points were given the same weight in the analysis. To allow for differences in quinolone doses in interaction studies, a dose of 1 mmol was chosen for all quinolones to compare the effects between substances. The bioavailability of a quinolone was taken into account by dividing the 1-mmol dose by the absolute oral bioavailability of the substance. For ciprofloxacin a value of 0.60 for oral bioavailability was used (20), for norfloxacin the value was 0.35 (1), and for all other compounds, it was 1.00 (2, 30, 33). Thus, the effect of a dose resulting in a bioavailable drug amount of 1 mmol was calculated from the fitted curves and was used to describe the Pin vivo of a quinolone. These doses (e.g., enoxacin, 321 mg; fleroxacin, 406 mg; ciprofloxacin, 553 mg) are within the range of therapeutic doses for most quinolones
(2, 30, 33). RESULTS Table 1 presents the Ein vitro values of the quinolones expressed as the remaining activity of P450IA2 for caffeine 3-demethylation when a quinolone (500 ,uM) was added to the incubation relative to the control activity in the absence of an inhibitor. Pin vitro values are also, given in Table 1. The coefficient of variation for Ein vitro between donors was small (about 10%). Enoxacin was the most effective inhibitor of caffeine 3-demethylation tested in vitro, whereas fleroxacin showed no inhibitory activity.
944
FUHR ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Pin vitro of quinolones by using caffeine 3-demethylation in human liver microsomes Compound
Enoxacin Ciprofloxacin Nalidixic acid Pipemidic acid Norfloxacin Lomefloxacin Pefloxacin Amifloxacin Difloxacin Ofloxacin Temafloxacin Fleroxacin
Ein *,tro determined with
liver microsomes from four donors (mean + SD)'
0.251 0.306 0.334 0.407 0.443 0.766 0.780 0.786 0.787 0.892 0.900 1.044
± ± ± ± ± ±
+ ± ± + + ±
0.017 0.067 0.056 0.028 0.089 0.071 0.048 0.038 0.073 0.099 0.045 0.142
Pin vitrob 0.749 0.694 0.666 0.593 0.557 0.234 0.220 0.214 0.213 0.108 0.100 0.000
a Expressed as the remaining activity of P450LA2 when a quinolone was added to the incubation as a fraction of the control activity. b Relative reduction of enzyme activity.
The Ein vivo values of the quinolones were assessed from the interaction studies with theophylline or caffeine reported above. The dose-dependent extent of the effects of the quinolones on caffeine metabolism were similar to those on theophylline degradation for all quinolones for which data from interaction studies were available. Whenever any effect of quinolones was found, higher doses caused a greater prolongation of the half-life, resulting in a higher Ein viv. (Table 2). Although study designs and study subjects differed widely, only small differences between the observed and the calculated effects were found for each quinolone (Table 2). Parameters estimated by using equation 2 for each quinolone are given in Table 3. As an example, the relation between the dose and inhibitory effect in vivo is shown for enoxacin and ciprofloxacin (Fig. 1). The extent of methylxanthine metabolism inhibition to be expected from the repeated application of a dose that results in a bioavailable quinolone amount of 1 mmol (i.e., 232 to 901 mg) (Pin viv.) was derived from reported values (see above) and is given in Fig. 2. The relationship of inhibitory potency in both test systems is shown by plotting Pin vv versus Pin vitro (Fig. 3). There was no linear relationship present for the substances tested here. However, by separating quinolone groups with high and low inhibitory potencies in vivo and in vitro by mean values, it was shown that pronounced decreases in cytochrome P450IA2 activity (Pin vitro, >0.39) in microsomes usually correspond to a marked prolongation of the methylxanthine half-life in vivo (Pin vivo, >0.22) and that minor effects in vitro were related only to slight changes in the pharmacokinetic parameters in volunteers. Exceptions were nalidixic acid, which had a high inhibitory effect in vitro but not in vivo, and pefloxacin, for which the effect in vivo was more pronounced than that in vitro. DISCUSSION
All quinolones tested (with the exception of fleroxacin) showed an inhibitory effect on caffeine 3-demethylation under the in vitro conditions used in this study. This effect was most pronounced for enoxacin and ciprofloxacin. A possible modification of the in vitro effects because of degradation of the quinolones has been tested for ciprofloxacin (12). Reduction in the concentration of intact ciproflox-
acin was not detectable as a result of incubation. The concentration of the Ml metabolite (cleavage of piperazinyl substituent) of ciprofloxacin was less than 0.3% of the ciprofloxacin concentration, and other metabolites were below the limit of detection. It has been reported that some quinolones (i.e., enoxacin, ciprofloxacin, ofloxacin, lomefloxacin, and pipemidic acid [14, 29]) exert a competitive type of inhibition on caffeine 3-demethylation. The structures of the substances tested previously (14, 29) cover the range of structures of those of the compounds included in the present investigation. Therefore, it is safe to conclude that all congeners used in this study exerted their inhibitory effects by binding to the active site of the enzyme, resulting in a competitive type of inhibition. A competitive inhibition is described by equation 3: v = (Vmax [S])/{[S] + Km (1 + [I]lKi)}, where v is the metabolite formation rate, Vm. is the maximum metabolite formation rate possible, [S] is the substrate concentration, Km is the Michaelis-Menten constant, [I] is the inhibitor concentration, and Ki is the inhibitor constant. By using equation 3, Ki values (or the 50% inhibitory concentration) can be calculated from a single substrate and inhibitor concentration since Km and Vm. values have previously been determined for this reaction in the same microsome samples (14). Therefore, the information given by the percent inhibition obtained in the present study is equivalent to that provided by the Ki or 50% inhibitory concentration parameters, which are used more often. However, the percentages were not transformed to avoid a multiplication of the experimental error. The low standard deviation between donors provides further evidence that P450 isozymes other than P450IA2 (or isoforms coordinately regulated with P450IA2) that are not susceptible to inhibition do not take part in caffeine metabolism (5). Cytochrome P450IA2 has been reported to be responsible for caffeine 3-demethylation, which is the major caffeine degradation step in humans (5). There is evidence that this isozyme also takes part in 1- and 7-demethylation of caffeine and mediates the main portions of the theophylline demethylation and hydroxylation pathways (although other cytochrome P450 isoforms may also play a minor part in primary theophylline metabolism [37]). Additionally, similar Km values for all primary metabolic steps of theophylline and caffeine have been reported (6, 14, 15, 21). Thus, it is not surprising that inhibition of P450IA2 activity by the concomitant application of quinolones results in a similar degree of half-life prolongation and/or clearance reduction for both caffeine and theophylline (10). This enables the pooling of in vivo inhibition data for both methylxanthines. The association between the increasing half-lives of the methylxanthines with higher doses of the inhibitory quinolones (16, 38) may readily be explained by the competitive type of inhibition that has been observed in vitro (14). To quantify the dose dependency of these inhibitory effects, we derived a dose- and bioavailability-normalized inhibitory potency (Pin vivo) for each quinolone for which interaction studies with methylxanthines have been reported previously. This defined a mean increase in the methylxanthine half-life that resulted from repeated, oral coadministration of a quinolone dose representing a bioavailable amount of 1 mmol. Pin vivo showed only minor variations within each of the compounds examined in this study, and therefore, it may be a good parameter to use to describe the inhibitory potency of quinolones. Nevertheless, each of the interaction studies
945
QUINOLONE-METHYLXANTHINE INTERACTION
VOL. 36, 1992
TABLE 2. Effects of quinolone antibacterial agents on methylxanthine elimination in vivo Quinolone administration Compound Compound
Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Enoxacin Pipemidic acid Pipemidic acid Pipemidic acid Pefloxacin Pefloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin
Ciprofloxacin Ciprofloxacin Ciprofloxacin Ciprofloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Norfloxacin Fleroxacin Fleroxacin Lomefloxacin Lomefloxacin Lomefloxacin Nalidixic acid Nalidixic acid Ofloxacin Ofloxacin Ofloxacin Ofloxacin Ofloxacin Temafloxacin
~
Compound (days) (days)'
25 mg b.i.d., 7 100 mg b.i.d., 7 400 mg b.i.d., 7 200 mg b.i.d., 4 100 mg b.i.d., 4 200 mg b.i.d., 4 400 mg b.i.d., 6 400 mg b.i.d., 7 500 mg b.i.d., 4 400 mg b.i.d., 4 400 mg b.i.d., 5 400 mg b.i.d., 4 800 mg b.i.d., 1.5 400 mg b.i.d., 6 400 mg b.i.d., 4 100 mg b.i.d., 4 250 mg b.i.d., 4 500 mg b.i.d., 4 750 mg b.i.d., 7 500 mg b.i.d., 7 750 mg b.i.d., 6 500 mg b.i.d., 7 500 mg b.i.d., 6 750 mg b.i.d., 1.5 400 mg b.i.d., 4 400 mg b.i.d., 4 200 mg t.i.d., 3 400 mg b.i.d., 6 800 mg b.i.d., 1.5 400 mg b.i.d., 7 400 mg b.i.d., 4 200 mg b.i.d., 5 400 mg b.i.d., 5 200 mg b.i.d., 5 400 mg b.i.d., 7 400 mg o.d., 5 500 mg b.i.d., 6 500 mg q.i.d., 7 200 mg b.i.d., 4 400 mg b.i.d., 6 200 mg t.i.d., 3 200 mg b.i.d., 5 200 mg b.i.d., 4 400 mg b.i.d., 7
Methylxanthine administration Dosing, duration Compound
Dosing, duration (days)
Theophylline Theophylline Theophylline Theophylline Caffeine Caffeine Theophylline Theophylline Caffeine Caffeine Theophylline Caffeine Caffeine Theophylline Caffeine Caffeine Caffeine Caffeine Theophylline Theophylline Theophylline Theophylline Theophylline Caffeine Theophylline Caffeine Theophylline Theophylline Caffeine Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Theophylline Caffeine Theophylline Theophylline Theophylline Theophylline Theophylline
200 mg, single dose 200 mg, single dose 200 mg, single dose 4 mg/kg, single dose 230 mg, single dose 230 mg, single dose 300-600 mg b.i.d. 146 mg b.i.d., 5 230 mg, single dose 183 mg o.d., 4 260 mg, single dose 230 mg, single dose 350 mg, single dose 300-600 mg b.i.d. 183 mg o.d., 4 230 mg, single dose 230 mg, single dose 230 mg, single dose 4 mg/kg, single dose 125 mg t.i.d., 7 200 mg b.i.d., 20 5 mg/kg, single dose 300-600 mg b.i.d. 100 mg, single dose 4 mg/kg, single dose 230 mg, single dose 183 mg, single dose 4 mg/kg, single dose 350 mg, single dose 5 mg/kg, single dose 142 mg t.i.d., 4 200 mg b.i.d., 9 141 mg b.i.d., 5 200 mg b.i.d., 10 125 mg t.i.d., 7 260 mg, single dose 300-600 mg b.i.d. 5 mg/kg, single dose 230 mg, single dose 300-600 mg b.i.d. 183 mg, single dose 200 mg t.i.d., 5 4 mg/kg, single dose 146 mg b.i.d., 5
E
iEn oba
eserved inE esimate
Reference
0.132 0.056 -0.247 -0.034 0.140 0.020 -0.085 -0.047 -0.024 0.136 -0.042 0.079 -0.039 -0.100 0.099 -0.005 -0.011 -0.035 -0.066 0.022 -0.052 0.048 0.066 0.040 -0.052 -0.002 0.068 0.008 -0.063 0.044 0.066 -0.033 0.017 -0.022 -0.026 0.037 -0.048 0.048 -0.031 -0.043 0.018 0.021 0.072 0.000
38 38 38 47 16 16 33 28 16 26 44 16 7 33 26 16 16 16 32 36 41 35 33 17 9 16 39 18 7 35 4 43 42 22 36 44 33 35 16 33 39
E.
0.259 0.424 0.452 0.504 0.508 0.558 0.614 0.652 0.720 0.835 0.427 0.548 0.600 0.291 0.490 0.056 0.128 0.209 0.260 0.266 0.274 0.292 0.310 0.366 0.084 0.136 0.144 0.149 0.184 0.185 0.207 0.000 0.081 0.000 0.018 0.081 0.000 0.096 0.000 0.017 0.049 0.052 0.103 0.086
11 47 28
a For calculation of parameters, see text; an Ein ,v, of 0 corresponds to no increase in the methylxanthine half-life during quinolone coadministration; of 1 represents a theoretical half-life prolongation to infinity. b o.d., once daily; b.i.d., two times daily; t.i.d., three times daily; q.i.d., four times daily. C Dose per kilogram of body weight.
(see above) showed
a
high interindividual variation in the
Ein ^i, value which may have been due to the well-known
intraindividual variation of methylxanthine metabolism in the course of a study and/or to interindividual differences in the disposition of quinolones. Differences in the susceptibilities of individual P450IA2 cytochromes in vivo seem to be of minor importance, since the effects of the quinolones on the enzyme in vitro were very similar between donors (Table 1). Thus, it is possible to estimate mean changes in methylxanthine kinetics because of quinolone coadministration at a defined dose by using equation 2 and the parameters listed in Table 3. For example, coadministration of pefloxacin (200 mg twice daily) in patients with asthma who are taking theophylline is estimated to cause an Ein i, of 0.243. This corresponds to a mean prolongation in the theophylline
a
value
TABLE 3. Parameters estimated by equation 2 describing the dose-effect relationship of the quinolones and inhibitory activity on methylxanthine elimination in vivo Compound
Enoxacin .........
Ciprofloxacin .........
ED50 (mg)a
172
1,550
Nalidixic acid ......... 9,917 452 Pipemidic acid ......... Norfloxacin ......... 2,441 Lomefloxacin ......... 8,760 624 Pefloxacin ......... Ofloxacin ......... 6,258 Temafloxacin ......... 4,251 Fleroxacin ......... 5,898 a The ED50 is the oral dose (given twice daily in most cases) estimated to double the caffeine or theophylline half-life when one of the quinolones is coadministered with one of the methylxanthines.
946
FUHR ET AL.
ANTIMICROB. AGENTS CHEMOTHER. 1.0& values reported for ciprofloxacin o values reported for enoxacin - curves fitted using eq. 2
0
>
*n
0.6
/
-4-,
13
0
c
13
0.8-
0.4
0
~~~~~~~~~~A 0f
2:N 0
C~~~~~~~~~~~~~C
0.01
10
100
1000
oral quinolone dose size
1 E+04
(mg)
FIG. 1. Influence of enoxacin and of ciprofloxacin dose on P450IA2 activity in vivo. The dose on the x axis describes the size of a single oral quinolone dose which was applied repeatedly. Ei i,. iS 1 minus the quotient of the methylxanthine half-lives reported without and during concomitant quinolone application. lE + 04, io'.
half-life of 32%, which requires a mean dose reduction by the same percentage to maintain steady-state trough levels when an open one-compartment model for theophylline kinetics is applied. However, a corresponding dose adjustment based on mean inhibitory effects in individual patients with longterm theophylline therapy still requires control for possible adverse drug reactions and/or concentrations of drug in serum because of interindividual diversity (see above). For the majority of quinolones investigated, the inhibitory
effects observed under the chosen in vitro conditions were related to qualitatively similar effects in vivo. Enoxacin, pipemidic acid, norfloxacin, and ciprofloxacin caused marked decreases in caffeine metabolite formation rates in vitro (Pin vitro .0.39) and produced a significant prolongation of methylxanthine half-lives in most in vivo studies (Pin viva' .0.22). Temafloxacin, lomefloxacin, ofloxacin, and fleroxacin had only minor effects both in vivo and in vitro. The inhibitory effects of quinolones on methylxanthine metabolism in vivo are postulated to be the result of a mutually exclusive concurrence at the cytochrome P450IA2
1.0-i 0
1.0-
0.8c
0~
0.8-
0~
0.6a.
0)
*ENX 0.6-
0. 0.40.
0.4-
a)
*PEF
_.
-0c
0.2-
*PPA
*NOR * CIp 0.2-
0.0-
FLXT 0.0
FIG. 2. Inhibitory potency of various quinolones against methylxanthine metabolism in vivo. Pi, ,j is 1 minus the quotient of the methylxanthine half-lives estimated without and during repeated application of a quinolone dose resulting in a bioavailable drug amount of 1 mmol. Abbreviations: ENX, enoxacin; PPA, pipemidic acid; PEF, pefloxacin; NOR, norfloxacin; CIP, ciprofloxacin; TEF, temafloxacin; FLX, fleroxacin; OFL, ofloxacin; LFX, lomefloxacin; NLA, nalidixic acid.
0.0
*LFX
*NLA
0.2 0.4 0.6 0.8 inh. potency in hum. liver microsomes (P in vitro)
1.0
FIG. 3. Relationship of inhibitory potency of quinolones against cytochrome P450IA2 activity in vivo and in vitro. Lines represent mean values that were used to distinguish between substances with minor inhibitory activities in vivo (Pi iv,a,
